The integrity of the genome is of prime importance to a dividing cell. Together, DNA repair and checkpoint responses ensure the integrity of the genome. Coordination of cell cycle checkpoints and DNA repair is especially important when unusually high loads of DNA damage are sustained following radiation or genotoxic chemotherapy. Mammalian Cds1 (also known as Chk2) is a checkpoint kinase that is activated in an ATM/ATR-dependent manner in response to DNA damage. In addition to delaying cell cycle progression, Cds1 homologs (Cds1 in fission yeast and Rad53 in budding yeast) have non-cell cycle functions that are important for survival following treatments that interrupt DNA replication or that damage DNA. Cds1 associates with a damage tolerance protein, Mus81, in fission yeast, implicating a direct role for Cds1 in DNA repair (Boddy et al., 2000, Molecular Cell Biol. 20:8758-66; hereinafter Boddy et al., 2000). In budding yeast, Mus81 mutants are reported to be sensitive to methyl methane sulfonate and to UV but not to agents that induce double-strand breaks (Interthal et al., 2000, Mol. Gen. Genet., 263:812-27; hereinafter Interthal, et al., 2000). Mus81 is important for survival following exposure to agents that block DNA replication, when DNA-polymerase function is compromised, and in the absence of the Bloom's syndrome helicase homologs (Rqh1 in fission yeast and Sgs1 in budding yeast, Boddy et al., 2000). These observations suggest a direct role for Mus81 in promoting recovery from problems encountered during replication.
In prokaryotes, reactivation of blocked replication forks is thought to proceed through a nonmutagenic pathway of homologous recombination. Several of the genes required for homologous recombination in vertebrate cells are essential for chromosomal stability. A number of genetic and physical observations suggest that Holliday junctions are intermediates in this recombination process (reviewed in Paques, et al., 1999, Microbiol. Mol. Biol. Rev., 63:349-404). Holliday junctions (HJs) are 4-stranded DNA crossover structures postulated as transient intermediates during genetic recombination and repair. Cleavage of the X-shaped HJs across an axis, performed by an HJ resolvase, is required to disentangle homologous duplexes. Recent studies suggest that HJs also arise at stalled replication forks (Seigneur et al., 1998, Cell, 95:419-30; hereinafter Seigneur et al., 1998). Thus, uncovering how HJs are resolved is vital for understanding mechanisms of genetic recombination, chromosomal replication, and genome maintenance.
Physical and genetic evidence for HJ formation exists from a number of different experimental systems. X-structures formed during meiosis have been observed in the budding yeast Saccharomyces cerevisiae (Collins, et al., 1994, Cell, 76:65-75). Evidence for replication-associated HJs was originally obtained with E. coli (Seigneur et al., 1998). These HJs are thought to form by the annealing of nascent strands at a stalled replication fork (known as fork regression). Evidence is mounting that HJs are an integral part of replication in eukaryotes. HJs accumulate at the rDNA locus during normal replication in S. cerevisiae, and this accumulation is enhanced by mutations in DNA replication polymerases α and δ (Zou et al., 1997, Cell, 90:87-96). X-structures were reported to form between sister chromatids during DNA replication in Physarum (Benard et al., 2001, Cell, 7:971-80; hereinafter Benard et al., 2001). Mutants of the fission yeast Schizosaccharomyces pombe that lack Rqh1 DNA helicase display enhanced mitotic recombination and are unable to segregate chromosomes when grown with the replication inhibitor hydroxyurea (Stewart et al., 1997, EMBO J, 16:2682-92). These phenotypes are partially rescued by expression of RusA, a bacterial HJ resolvase, indicating that Rqh1 may be involved in branch migration of HJs that arise at regressed replication forks (Doe et al., 2000, EMBO J, 19:2751-62; hereinafter Doe et al., 2000).
The best characterized HJ resolvase is RuvC of E. coli, which is part of the RuvABC complex that branch migrates and cleaves HJs (Bennett et al., 1993, Cell, 74: 1021-1031). Interestingly, there are no known eukaryotic sequence counterparts of bacterial resolvases, although eukaryotes have mitochondrial HJ resolvases that may be ancestrally related to RuvC (Lilley et al., 2001, Nat. Rev. Mol. Cell Biol., 2:433-43 hereinafter Lilley et al., 2001). Recent studies suggest that HJ branch migration and resolvase activities may associate in calf testes and mammalian cell lines (Constantinou et al., 2001, EMBO Rep., 1:80-84), but eukaryotic nuclear HJ resolvases have thus far eluded identification.
The ERCC1-XPF family of heterodimeric enzymes constitute another interesting class of structure-specific endonucleases. ERCC1-XPF, which has no bacterial orthologs, cuts duplex DNA with a defined polarity on the 5′ side of a junction between double-strand and single-strand DNA (Sijbers et al., 1996, Cell, 86:811-22). ERCC1-XPF is essential for nucleotide excision repair (NER), where it incises the damaged strand on the 5′ side of the lesion. The ERCC1-XPF nuclease family also appears to participate in various recombination pathways (Paques, et al., 1999, Microbiol. Mol. Biol. Rev., 63: 349-404). For example, in Drosophilia melanogaster, MEI-9, an XPF homolog, is required for normal levels of meiotic recombination (Sekelsky et al., 1995, Genetics, 141:619-27).
Mus81, a novel XPF-related protein, was recently discovered through its association with the replication checkpoint kinase Cds1 in fission yeast and the recombination repair protein Rad54 in budding yeast (Boddy et al., 2000; Interthal et al., 2000). Strikingly, fission yeast Mus81 cells exhibit phenotypes expected of an HJ resolvase mutant (Boddy et al., 2000). Mus81 is important for cell viability in a variety of circumstances that impede replication fork progression, such as unrepaired thymine dimers, nucleotide starvation, and compromised DNA polymerase alleles. Mus81 is essential in Rqh1 cells of fission yeast, which are thought to accumulate HJs during DNA replication (Doe et al., 2000). Moreover, Mus81 is required for production of viable spores, a process that is thought to depend on HJ resolution prior to meiosis I (Boddy et al., 2000; Interthal et al., 2000). Mus81 is also involved in resolution of HJs (Boddy et al., 2000).
Boddy et al., 2001, Cell 107: 537-548 (hereinafter Boddy et al., 2001), have reported that the endonuclease activity of Mus81 in fission yeast depends upon the presence of a particular binding partner, essential meiotic endonuclease 1 (Eme1). Thus both Mus81 and Eme1 are subunits of an endonuclease complex, which is analogous to the well characterized endonuclease ERCC1-XPF. Boddy et al. also reported that Eme1 has no sequence homology with ERCC1, whereas Mus81 shares homology with the C-terminus of XPF (Boddy et al., 2001). Mus81 and Eme1 are reported to interact through their C-termini.
Chen et al. have reported that the human homolog of Mus81 (Hmus81) has endonuclease activity and cleaves Holliday Junctions in vivo (Chen et al., 2001, Molecular Cell, 8:1117-1127; hereinafter Chen et al., 2001). A number of murine homologs of Mus81 (Mmus81) are disclosed in U.S. Pat. No. 6,440,732 to Russell et al.
In humans, excision repair is an important defense mechanism against two major carcinogens: sunlight and cigarette smoke. It has been found that individuals defective in excision repair exhibit a high incidence of cancer (Sancar, 1996, “DNA Excision Repair” Ann. Rev. Biochem. 65:43-81). Other mechanisms are also available for DNA repair, such as mismatch repair, which stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. Inactivation of genes encoding enzymes involved in these repair mechanisms reportedly result in a large increase in spontaneous mutability and a predisposition to tumor development. (Modrich et al., 1996, “Mismatch Repair in Replication Fidelity, Genetic Recombination and Cancer Biology” Ann. Rev. Biochem. 65:101-33). The importance of maintaining genomic fidelity is amply illustrated by the many available mechanisms for repair, and if unrepairable, by the arrest of cell division. (Wood, 1996, “DNA Repair in Eukaryotes” Ann. Rev. Biochem. 65:135-67).
Many chemotherapeutic agents are designed to disrupt or otherwise cause damage to the DNA of targeted malignant cells. Antineoplastic agents such as alkylating agents, antimetabolites, and other chemical analogs and substances typically act by inhibiting nucleotide biosynthesis or protein synthesis, cross-linking DNA, or intercalating with DNA to inhibit replication or gene expression. Bleomycin and etoposide, for example, specifically damage DNA and prevent repair.
The inhibition of DNA damage repair activity amplifies the potency of antineoplastic agents, and enhances the efficacy of their use as chemotherapeutic agents. For example, the targeted cells are relatively more susceptible to damage caused by chemotherapeutic agents when repair mechanisms are inhibited, so that reduced dosages of the chemotherapeutic agents can be used, in proportion to the increased efficacy, thus reducing unwanted side effects.
Diseases can also result from defective DNA repair mechanisms, including, for example, hereditary nonpolyposis colorectal cancer (defect in mismatch repair), Nijmegen breakage syndrome (defect in double strand break repair), Xeroderma pigmentosum, Cockayne syndrome, and Trocothiodystrophy (defects in nuclear excision repair), and the like (Lengauer et al., 1998, “Genetic instabilities in human cancers” Nature, 396(6712):643-649; Kanaar et al., 1998, “Molecular mechanisms of DNA double stranded repair” Trends Cell Biol. 8(12):483489).
It is further envisioned that the transient inhibition of DNA checkpoint and DNA damage arrest in dividing cells may allow the use of relatively lower doses of chemotherapeutic agents to effect relatively greater damage to targeted cells in the treatment of diseases such as cancer.